The long-term goal of this laboratory is to use cellular and molecular methodologies to study the organization and expression of human chromosomes and their constituent genes and regulatory sequences in normal and disease states. The specific aim of this component proposal is to use somatic cell genetics, molecular probes, microdissection and microcloning techniques for fine structure mapping, dissection and expression of human chromosome 21. These studies will provide direct link bridging cytogenetic and molecular resolution of chromosome 21 and can facilitate our search for genes and regulatory sequences underlying the etiology of Down syndrome. The proposed studies include (1) mapping of chromosome 21 using a series of well-defined terminal deletions, (2) microcloning of DNA sequences from defined areas of chromosome 21, and (3) combined use of microdissection and microinjection for studying expression of large blocks of DNA in cultured cells and animals. For terminal deletion mapping, we will establish large numbers of terminal deletions of chromosome 21 by using a human/CHO cell hybrid containing a 21q+ translocation chromosome derived from AML-M2 leukemia cells of t(8;21)(q22;q22.3). Terminal deletions will be produced by X-ray and selected by BrdU + visible light method using the auxotrophic marker GlyB which was translocated from chromosome 8 to 21 of the 21q+ translocation chromosome. Large numbers of DNA probes will be used to define the deletions in linear and submicroscopic resolution. These deletions will be used for refined regional mapping of all genes and DNA probes assigned to chromosome 21, and for resolving the order of many closely linked genes and DNA markers on this chromosome. For microdissecting chromosome 21, we will use the newly developed microdissection and microcloning techniques. Chromosome 21 can be subdivided into five regions and region-specific libraries will be constructed for providing DNA probes from defined areas of the chromosome for possible identification of DNA sequences associated with Down syndrome. Finally we will introduce large blocks of contiguous DNA sequences from dissected regions of chromosome 21 into cells by microinjection for studying their integration and expression. This work will be extended to transgenic mice. This approach appears particularly important for studying expression of complex disorders like Down syndrome.